Investigations will be carried out upon the endopeptidase, carboxypeptidase, and aminopeptidase components of Pronase. Work has been initiated and will be continued towards determining the primary structure of aminopeptidase-1. This enzyme is the smallest known aminopeptidase. The aminopeptidase will also be applied as an aid in determining the primary structure of substrate polypeptide chains. Since the aminopeptidase has no prolidase activity its action stops when proline is present at the penultimate site. As we have already described this should provide smaller peptides from a parent molecule, having new starting points for automated sequence analysis. Studies on the carboxypeptidase will be extended so as to determine that some sequence of the polypeptide chain shows homology with published sequences for the mammalian carboxypeptidases. This will confirm the very strong evidence already noted for homology. Other studies will include the establishment of the sites of acetylation with acetic anhydride of guanidine-stable chymoelastase. In the absence of glycerol this enzyme is acetylated with complete loss of activity. In the presence of glycerol the enzyme can be acetylated with all amino groups blocked and complete retention of activity. The determination of the sites of acetylation which inactivate the enzyme will contribute to further knowledge regarding structure-function relationships of this enzyme. Studies will be extended to determine the higher-molecular-weight components which are present in Pronase. Since these components have proteolytic activity their specificities will be determined after purification. BIBLIOGRAPHIC REFERENCES: Seber, J.F., Toomey, T.P., Powell, J.T., Brew, K., and Awad, W.M., Jr. (1976) J. Biol. Chem. 251, 204. The Proteolytic Enzymes of the K-1 Strain of Streptomyces griseus Obtained from a Commercial Preparation (Pronase). Purification and Characterization of the Carboxypeptidase. Vosbeck, K.D., Greenberg, B.D., Ochoa, M.S., Whitney, P.L., and Awad, W.M., Jr. (1976) Fed. Proc. 35, 1474. Effect of pH, Metal Ions, and Amino Acids on Pronase Aminopeptidase.